Our goal is to integrate systems biology approaches into the analysis of cancer drug resistance mechanisms, and thus to develop and evaluate new strategies for the efficient selection of optimal therapeutic combinations. In our recent work, we used siRNA screening to interrogate over 600 candidate genes from a set we nominated by network modeling to be enriched for regulators of EGFR signaling. This and subsequent analysis identified SC4MOL (sterol C4-methyl oxidase-like), a little-studied intermediate in the sterol biosynthesis pathway, as a potent regulator of cell viability following treatment with the EGFR inhibitors erlotinib and cetuximab. To understand the mechanism of SC4MOL action, we again used a bioinformatics strategy to model an interaction network for evolutionarily conserved orthologs of SC4MOL and its partner protein, NSDHL (NADP-dependent steroid dehydrogenase-like). This predicted multiple connections to regulators of exocytic trafficking and protein degradation. In direct preliminary test of the network predictions, we demonstrated that RNAi depletion of SC4MOL or NSDHL resulted in loss of EGFR from the plasma membrane, and enhanced its ubiquitination and degradation. Our hypothesis is that loss of interactions between SC4MOL and NSDHL with partner proteins influences trafficking so as to accelerate EGFR degradation, thereby potentiating EGFR-targeting drugs. To further validate this approach to identifying modifiers of targeted response, and potentially validate SC4MOL and NSDHL as biomarkers of drug response and/or targets for drug development, we will perform the following two Aims: Aim 1. Investigate the mechanism by which SC4MOL and NSDHL regulate EGFR trafficking. We will assess how SC4MOL and NSDHL influence EGFR trafficking and degradation, and whether the catalytic activity of SC4MOL and NSDHL is required for their actions. We will analyze interactions between SC4MOL and a small set of predicted conserved interaction partners with known roles in regulating vesicular trafficking predicted by bioinformatics analysis of the orthologs in lower eukaryotes. Aim 2. Investigate the interaction of SC4MOL and NSDHL with ERAD proteins. We have found silencing of SC4MOL or NSDHL enhances EGFR ubiquitination and lysosomal degradation. Our analysis identified a number of candidates within the ER-associated protein degradation (ERAD) system, which will be tested for physical association with SC4MOL and NSDHL, and for a role in SC4MOL- and NSDHL-dependent regulation of EGFR expression, trafficking, and degradation. Given the prominence of EGFR as a target in cancer therapy, this work has potentially high impact to reduce drug resistance relevant to many clinically valuable therapeutic agents.